Skip to main content

Advertisement

SpringerLink
Log in

pHLIP Peptides Target Acidity in Activated Macrophages

  • Research Article
  • Published:
Molecular Imaging and Biology Aims and scope Submit manuscript

Abstract

Purpose

Acidity can be a useful alternative biomarker for the targeting of metabolically active cells in certain diseased tissues, as in acute inflammation or aggressive tumors. We investigated the targeting of activated macrophages by pH low insertion peptides (pHLIPs), an established technology for targeting cell-surface acidity.

Procedures

The uptake of fluorescent pHLIPs by activated macrophages was studied in cell cultures, in a mouse model of lung inflammation, and in a mouse tumor model. Fluorescence microscopy, whole-body and organ imaging, immunohistochemistry, and FACS analysis were employed.

Results

We find that cultured, activated macrophages readily internalize pHLIPs. The uptake is higher in glycolytic macrophages activated by LPS and INF-γ compared to macrophages activated by IL-4/IL-13. Fluorescent pHLIPs target LPS-induced lung inflammation in mice. In addition to marking cancer cells within the tumor microenvironment, fluorescent pHLIPs target CD45+, CD11b+, F4/80+, and CD206+ tumor-associated macrophages with no significant targeting of other immune cells. Also, fluorescent pHLIPs target CD206-positive cells found in the inguinal lymph nodes of animals inoculated with breast cancer cells in mammary fat pads.

Conclusions

pHLIP peptides sense low cell surface pH, which triggers their insertion into the cell membrane. Unlike cancerous cells, activated macrophages do not retain inserted pHLIPs on their surfaces, instead their highly active membrane recycling moves the pHLIPs into endosomes. Targeting activated macrophages in diseased tissues may enable clinical visualization and therapeutic opportunities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Parisi L, Gini E, Baci D et al (2018) Macrophage polarization in chronic inflammatory diseases: killers or builders? J Immunol Res 2018:8917804

    PubMed  PubMed Central  Google Scholar 

  2. Davies LC, Jenkins SJ, Allen JE, Taylor PR (2013) Tissue-resident macrophages. Nat Immunol 14:986–995

    PubMed  PubMed Central  Google Scholar 

  3. Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723–737

    PubMed  PubMed Central  Google Scholar 

  4. Murray PJ, Allen JE, Biswas SK et al (2014) Macrophage activation and polarization: nomenclature and experimental guidelines. Immunity 41:14–20

    PubMed  PubMed Central  Google Scholar 

  5. Geeraerts X, Bolli E, Fendt SM, Van Ginderachter JA (2017) Macrophage metabolism as therapeutic target for cancer, atherosclerosis, and obesity. Front Immunol 8:289

    PubMed  PubMed Central  Google Scholar 

  6. Viola A, Munari F, Sanchez-Rodriguez R, Scolaro T, Castegna A (2019) The metabolic signature of macrophage responses. Front Immunol 10:1462

    PubMed  PubMed Central  Google Scholar 

  7. Vitale I, Manic G, Coussens LM, Kroemer G, Galluzzi L (2019) Macrophages and metabolism in the tumor microenvironment. Cell Metab 30:36–50

    PubMed  Google Scholar 

  8. Pan Y, Yu Y, Wang X, Zhang T (2020) Tumor-associated macrophages in tumor immunity. Front Immunol 11:583084

    PubMed  PubMed Central  Google Scholar 

  9. Warburg O, Wind F, Negelein E (1927) The metabolism of tumors in the body. J Gen Physiol 8:519–530

    PubMed  PubMed Central  Google Scholar 

  10. Covarrubias AJ, Aksoylar HI, Yu J, et al. (2016) Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation. Elife 5.

  11. Penny HL, Sieow JL, Gun SY, et al. (2021) Targeting glycolysis in macrophages confers protection against pancreatic ductal adenocarcinoma. Int J Mol Sci 22.

  12. Chiba S, Hisamatsu T, Suzuki H et al (2017) Glycolysis regulates LPS-induced cytokine production in M2 polarized human macrophages. Immunol Lett 183:17–23

    PubMed  Google Scholar 

  13. Wu H, Yin Y, Hu X et al (2019) Effects of environmental pH on macrophage polarization and osteoimmunomodulation. ACS Biomater Sci Eng 5:5548–5557

    PubMed  Google Scholar 

  14. El-Kenawi A, Gatenbee C, Robertson-Tessi M et al (2019) Acidity promotes tumour progression by altering macrophage phenotype in prostate cancer. Br J Cancer 121:556–566

    PubMed  PubMed Central  Google Scholar 

  15. Genard G, Lucas S, Michiels C (2017) Reprogramming of tumor-associated macrophages with anticancer therapies: radiotherapy versus chemo- and immunotherapies. Front Immunol 8:828

    PubMed  PubMed Central  Google Scholar 

  16. Wenes M, Shang M, Di Matteo M et al (2016) Macrophage metabolism controls tumor blood vessel morphogenesis and metastasis. Cell Metab 24:701–715

    PubMed  Google Scholar 

  17. Wu H, Estrella V, Beatty M et al (2020) T-cells produce acidic niches in lymph nodes to suppress their own effector functions. Nat Commun 11:4113

    PubMed  PubMed Central  Google Scholar 

  18. Loike JD, Silverstein SC (1983) A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages. J Immunol Methods 57:373–379

    PubMed  Google Scholar 

  19. Anderson M, Moshnikova A, Engelman DM, Reshetnyak YK, Andreev OA (2016) Probe for the measurement of cell surface pH in vivo and ex vivo. Proc Natl Acad Sci U S A 113:8177–8181

    PubMed  PubMed Central  Google Scholar 

  20. Li C, Levin M, Kaplan DL (2016) Bioelectric modulation of macrophage polarization. Sci Rep 6:21044

    PubMed  PubMed Central  Google Scholar 

  21. Crawford T, Moshnikova A, Roles S et al (2020) pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery. Sci Rep 10:18356

    PubMed  PubMed Central  Google Scholar 

  22. Wyatt LC, Lewis JS, Andreev OA, Reshetnyak YK, Engelman DM (2017) Applications of pHLIP technology for cancer imaging and therapy. Trends Biotechnol 35:653–664

    PubMed  PubMed Central  Google Scholar 

  23. Dharmaratne NU, Kaplan AR, Glazer PM (2021) Targeting the hypoxic and acidic tumor microenvironment with pH-sensitive peptides. Cells 10.

  24. Adochite RC, Moshnikova A, Golijanin J, Andreev OA, Katenka NV, Reshetnyak YK (2016) Comparative study of tumor targeting and biodistribution of pH (low) insertion peptides (pHLIP((R)) peptides) conjugated with different fluorescent dyes. Mol Imaging Biol 18:686–696

    PubMed  PubMed Central  Google Scholar 

  25. Adochite RC, Moshnikova A, Carlin SD et al (2014) Targeting breast tumors with pH (low) insertion peptides. Mol Pharm 11:2896–2905

    PubMed  PubMed Central  Google Scholar 

  26. Moshnikova A, Moshnikova V, Andreev OA, Reshetnyak YK (2013) Antiproliferative effect of pHLIP-amanitin. Biochemistry 52:1171–1178

    PubMed  Google Scholar 

  27. Reshetnyak YK, Andreev OA, Lehnert U, Engelman DM (2006) Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix. Proc Natl Acad Sci U S A 103:6460–6465

    PubMed  PubMed Central  Google Scholar 

  28. Reshetnyak YK, Segala M, Andreev OA, Engelman DM (2007) A monomeric membrane peptide that lives in three worlds: in solution, attached to, and inserted across lipid bilayers. Biophys J 93:2363–2372

    PubMed  PubMed Central  Google Scholar 

  29. Andreev OA, Karabadzhak AG, Weerakkody D, Andreev GO, Engelman DM, Reshetnyak YK (2010) pH (low) insertion peptide (pHLIP) inserts across a lipid bilayer as a helix and exits by a different path. Proc Natl Acad Sci U S A 107:4081–4086

    PubMed  PubMed Central  Google Scholar 

  30. Hunt JF, Rath P, Rothschild KJ, Engelman DM (1997) Spontaneous, pH-dependent membrane insertion of a transbilayer alpha-helix. Biochemistry 36:15177–15192

    PubMed  Google Scholar 

  31. Reshetnyak YK (2008) Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane. Chim Oggi 105:15340–15345

    Google Scholar 

  32. Kyrychenko A, Vasquez-Montes V, Ulmschneider MB, Ladokhin AS (2015) Lipid headgroups modulate membrane insertion of pHLIP peptide. Biophys J 108:791–794

    PubMed  PubMed Central  Google Scholar 

  33. Vasquez-Montes V, Gerhart J, King KE, Thevenin D, Ladokhin AS (2018) Comparison of lipid-dependent bilayer insertion of pHLIP and its P20G variant. Biochim Biophys Acta Biomembr 1860:534–543

    PubMed  Google Scholar 

  34. Karabadzhak AG, Weerakkody D, Deacon J, Andreev OA, Reshetnyak YK, Engelman DM (2018) Bilayer thickness and curvature influence binding and insertion of a pHLIP peptide. Biophys J 114:2107–2115

    PubMed  PubMed Central  Google Scholar 

  35. Westerfield J, Gupta C, Scott HL et al (2019) Ions modulate key interactions between pHLIP and lipid membranes. Biophys J 117:920–929

    PubMed  PubMed Central  Google Scholar 

  36. Schlebach JP (2019) Ions at the interface: pushing the pK of pHLIP. Biophys J 117:793–794

    PubMed  PubMed Central  Google Scholar 

  37. Vasquez-Montes V, Gerhart J, Thevenin D, Ladokhin AS (2019) Divalent cations and lipid composition modulate membrane insertion and cancer-targeting action of pHLIP. J Mol Biol 431:5004–5018

    PubMed  PubMed Central  Google Scholar 

  38. Gupta C, Ren Y, Mertz B (2018) Cooperative nonbonded forces control membrane binding of the pH-low insertion peptide pHLIP. Biophys J 115:2403–2412

    PubMed  PubMed Central  Google Scholar 

  39. Brown MC, Yakubu RA, Taylor J et al (2014) Bilayer surface association of the pHLIP peptide promotes extensive backbone desolvation and helically-constrained structures. Biophys Chem 187-188:1–6

    PubMed  Google Scholar 

  40. Scott HL, Heberle FA, Katsaras J, Barrera FN (2019) Phosphatidylserine asymmetry promotes the membrane insertion of a transmembrane helix. Biophys J 116:1495–1506

    PubMed  PubMed Central  Google Scholar 

  41. Li N, Yin L, Thevenin D et al (2013) Peptide targeting and imaging of damaged lung tissue in influenza-infected mice. Future Microbiol 8:257–269

    PubMed  Google Scholar 

  42. Price NL, Miguel V, Ding W, et al. (2019) Genetic deficiency or pharmacological inhibition of miR-33 protects from kidney fibrosis. JCI Insight 4.

  43. Andreev OA, Dupuy AD, Segala M et al (2007) Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo. Proc Natl Acad Sci U S A 104:7893–7898

    PubMed  PubMed Central  Google Scholar 

  44. Henry KE, Chaney AM, Nagle VL, et al. (2020) Demarcation of sepsis-induced peripheral and central acidosis with pH-low insertion cyclic (pHLIC) peptide. J Nucl Med.

  45. Sahraei M, Chaube B, Liu Y et al (2019) Suppressing miR-21 activity in tumor-associated macrophages promotes an antitumor immune response. J Clin Invest 129:5518–5536

    PubMed  PubMed Central  Google Scholar 

  46. Taylor PR, Martinez-Pomares L, Stacey M, Lin HH, Brown GD, Gordon S (2005) Macrophage receptors and immune recognition. Annu Rev Immunol 23:901–944

    PubMed  Google Scholar 

  47. Allavena P, Chieppa M, Bianchi G et al (2010) Engagement of the mannose receptor by tumoral mucins activates an immune suppressive phenotype in human tumor-associated macrophages. Clin Dev Immunol 2010:547179

    PubMed  Google Scholar 

  48. Scodeller P, Simon-Gracia L, Kopanchuk S et al (2017) Precision targeting of tumor macrophages with a CD206 binding peptide. Sci Rep 7:14655

    PubMed  PubMed Central  Google Scholar 

  49. Zhang C, Yu X, Gao L et al (2017) Noninvasive imaging of CD206-positive M2 macrophages as an early biomarker for post-chemotherapy tumor relapse and lymph node metastasis. Theranostics 7:4276–4288

    PubMed  PubMed Central  Google Scholar 

  50. Kurahara H, Takao S, Maemura K et al (2013) M2-polarized tumor-associated macrophage infiltration of regional lymph nodes is associated with nodal lymphangiogenesis and occult nodal involvement in pN0 pancreatic cancer. Pancreas 42:155–159

    PubMed  Google Scholar 

  51. Wang Y, Wang J, Yang C et al (2021) A study of the correlation between M2 macrophages and lymph node metastasis of colorectal carcinoma. World J Surg Oncol 19:91

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful for the support of Stefania Andreev for making graphics for Fig. 6, and Dr. Dhammika Weerakkody for support and maintenance of the laboratory at URI.

Funding

This work was supported by NIH grant R01 GM073857 (Y.K.R., O.A.A., and D.M.E.). Members of the URI Institutional Development Award (IDeA) Network for Biomedical Research Excellence received a support from NIH P20 GM103430.

Author information

Author notes

  1. Hannah Visca and Michael DuPont contributed equally.

Authors and Affiliations

  1. Physics Department, University of Rhode Island, Kingston, RI, USA

    Hannah Visca, Michael DuPont, Anna Moshnikova, Troy Crawford, Oleg A. Andreev & Yana K. Reshetnyak

  2. Department of Molecular Biophysics and Biochemistry, Yale, New Haven, CT, USA

    Donald M. Engelman

Authors
  1. Hannah Visca
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael DuPont
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anna Moshnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Troy Crawford
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Donald M. Engelman
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Oleg A. Andreev
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yana K. Reshetnyak
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

O.A.A. and Y.K.R. designed research; H.V., M.D., A.M., and T.C. performed research; H.V., M.D., A.M., T.C., and Y.R. analyzed data; and D.M.E., O.A.A., and Y.K.R. wrote the paper. All authors read, provided feedback on, and approved the manuscript for publication. H.V. and M.D. equally contributed to the work.

Corresponding author

Correspondence to Yana K. Reshetnyak.

Ethics declarations

Conflict of Interest

D.M.E., O.A.A., and Y.K.R. are founders of pHLIP, Inc., and they have shares in the company. pHLIP, Inc provided funding for the manufacturing of pHLIP ICG, the FACS analysis performed at the Charles River Discovery Labs, and the in vivo portion of the study for targeting of inflamed lungs in mice performed at Melior Discovery.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

ESM 1

(DOCX 1335 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Visca, H., DuPont, M., Moshnikova, A. et al. pHLIP Peptides Target Acidity in Activated Macrophages. Mol Imaging Biol 24, 874–885 (2022). https://doi.org/10.1007/s11307-022-01737-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11307-022-01737-x

Key words

Search

Navigation